Targeting Human MicroRNA Genes Using Engineered Tal-Effector Nucleases (TALENs)

<div><p>MicroRNAs (miRNAs) have quickly emerged as important regulators of mammalian physiology owing to their precise control over the expression of critical protein coding genes. Despite significant progress in our understanding of how miRNAs function in mice, there remains a fundamental need to be able to target and edit miRNA genes in the human genome. Here, we report a novel approach to disrupting human miRNA genes <i>ex vivo</i> using engineered TAL-effector (TALE) proteins to function as nucleases (TALENs) that specifically target and disrupt human miRNA genes. We demonstrate that functional TALEN pairs can be designed to enable disruption of miRNA seed regions, or removal of entire hairpin sequences, and use this approach to successfully target several physiologically relevant human miRNAs including miR-155*, miR-155, miR-146a and miR-125b. This technology will allow for a substantially improved capacity to study the regulation and function of miRNAs in human cells, and could be developed into a strategic means by which miRNAs can be targeted therapeutically during human disease.</p></div

Using two TALEN pairs to delete the entire human miR-155 hairpin sequence.

<p>(A) TALEN pairs targeting miR-155 were designed and constructed (called TALEN C). The upper panel shows a schematic of the TALEN A pair binding sites. The lower panel shows the sequence alignments comparing Wt and TALEN C mutated miR-155. The left and right TALEN binding sites are highlighted in yellow and the miR-155 seed region is boxed in red. (B) Schematic of the binding sites of two TALEN pairs (TALEN A and TALEN C) targeting miR-155. (C–D) Both TALEN A and TALEN C pairs were transfected into 293T cells. The miR-155 locus was amplified by PCR and subjected to TOPO cloning and sequencing. (C) Electrophoresis gel analysis showing deletions in the miR-155 locus. The arrows on the right indicate the two expected PCR products with or without large deletions. (D) Sequence alignments between a Wt clone and two TOPO clones with large deletions. The left and right TALEN binding sites for both TALEN A and TALEN C are highlighted in yellow and the miR-155 hairpin sequence is boxed in red.</p

Using TALENs to target the seeds of human miR-146a and miR-125b1.

<p>TALEN pairs targeting the indicated miRNAs were designed and constructed. After the TALEN pairs were transfected into 293T cells, methods were performed (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063074#pone-0063074-g002" target="_blank">Figure 2</a>) to detect mutations in the targeted regions. (A, B) The upper panel shows the schematics of the TALEN pair binding sites adjacent to (A) miR-146a and (B) miR-125b1. The lower panel shows the sequence alignments between the Wt and mutated miRNA genes. The left and right TALEN binding sites are highlighted in yellow and the miRNA seed regions are boxed in red.</p

The miR-155* targeting TALEN pair causes mutations in the miR-155* sequence.

<p>293T cells were transfected with or without plasmids encoding the TALEN pair designed to target the miR-155* region. A GFP expressing plasmid was co-transfected. (A) 48 hours later, GFP+ cells were sorted by FACS and subjected to gDNA extraction. (B–F) The miR-155* targeted region was amplified by PCR and subjected to an HRMA analysis (B–D) or TOPO cloning and sequencing (E,F). (B) Schematic of the HRMA approach. A small region of the genome that includes different lengths of DNA deletions is amplified by PCR. Upon annealing, different types of homoduplex and heteroduplex dsDNA molecules are produced with different melting temperatures. (C) HRMA of the mir-155* PCR amplicons generated using gDNA from Wt (mock transfected 293t cells), Unsorted (293t cells with the TALEN pair transfection), Sorted GFP+ and sorted GFP-(293t cells with the TALEN pair and a GFP plasmid co-transfection followed by FACS sorting). (D) The results of the HRMA analysis are also shown as fluorescence difference plots using the normalized data. The Wt sample is used as the baseline. (E) PCR products from C were cloned into a TOPO vector and the length of the individual DNA fragment was assessed by gel electrophoresis. (F) Sequencing results of TOPO clones from E are shown. They are aligned with the wild-type miR-155* sequence. The left and right TALEN binding sites are highlighted in yellow and the miR-155* region is boxed in red.</p

Schematic of the miR-155/miR-155* genomic locus and the location of the TALEN pair engineered to target the miR-155* region.

<p>(A) Schematic of the miR-155/miR-155* genomic locus. The three BIC exons are shown in yellow and the miR-155 hairpin is in red. (B) Schematic of the miR-155 hairpin structure. The miR-155 arms are shown in black, while the mature miR-155 and miR-155* sequences are in dark grey. Blue and green boxes represent the binding sites of the TALEN pair designed to target the miR-155* region, and hexagons represent the heterodimerized FokI enzyme positioned over the spacer sequence. (C) The two expression plasmids containing the TALEN pair along with the FokI nuclease domain, and the TALEN-RVD sequences corresponding to each targeted DNA sequence are shown. The details of pCS2TAL3-DDD and pCS2TAL3-RRR expression vectors are described in the Materials and Methods section. NN, HD, NG and NI represent the RVD regions of each repeat sequence that bind to nucleotide G, C, T and A, respectively. The left and right TALEN binding sequences are shown in red and purple, respectively, and the spacer region is in blue.</p

The miR-155* targeting TALEN pair causes both bi-allelic and mono-allelic mutations in human cells.

<p>(A) Schematic of the experimental design. 293T cells were transfected with the TALEN pair targeting the miR-155* region along with a GFP plasmid. 48 hours later, GFP+ cells were sorted by FACS and subjected to single cell cloning. After individual cell clones were expanded, gDNA was extracted from the cell clones. The miR-155* TALEN pair-targeted region was amplified by PCR and subjected to TOPO cloning and sequencing. (B,C) Representative cell clones showing bi-allelic mutations (B) or mono-allelic mutations (C). In the sequence alignment graph, the left and right TALEN binding sites are highlighted in yellow and the miR-155* region is in the red box.</p

Mutation rate mediated by TALEN pairs in transfected 293T cells.

<p>Mutation rate mediated by TALEN pairs in transfected 293T cells.</p

Simple Methods for Generating and Detecting Locus-Specific Mutations Induced with TALENs in the Zebrafish Genome

<div><p>The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line mutations at a pre-specified locus.</p> </div

Dose-dependent induction of mutations with TALENs.

<p>WT embryos were either not injected or injected at the 1 cell stage with 4 pg, 20 pg, or 100 pg total <i>gol-ex2</i> TALEN RNA. gDNA was prepared from individual 1 dpf embryos and analyzed by HRMA for the presence of targeted mutations. Every embryo injected with 20 or 100 pg total RNA had mutations, but some 4 pg RNA-injected embryos did not have clear evidence of induced mutations (dark blue). As the amount of injected RNA was increased, the melt profiles of injected embryos, on average, diverged increasingly from the WT curves, indicating that increasing amounts of TALENs produce increasing numbers of mutant genomes.</p

Induction of germ line mutations with TALENs.

<p>(A) TALEN RNA-injected G0 founders transmit targeted mutations to their offspring. G0 founders were mated with WT partners and the gDNAs of individual 1 dpf F1 progeny were examined by HRMA for the presence of mutations. The region flanking targeted sites was cloned and sequenced following PCR amplification from the gDNA of F1 offspring. Examples of germ line-transmitted mutations at each targeted locus are shown (purple and pink boxed sequences indicate Left and Right RVD binding sites of the TALENs, respectively; red indicates sequence alterations). (B, C) The germ lines of individual G0 founders are mosaic and can harbor several targeted mutations. Sibling F1 progeny of a single G0 founder targeted at <i>gol</i> (B) or a single G0 founder targeted at <i>ryr3</i> (C) were examined by HRMA for inheritance of new mutations at <i>gol</i> or <i>ryr3</i>, respectively. Some F1 embryos (<i>+/+</i>) had no mutations, as their HRMA profiles (blue) resembled that of control WT embryos (grey). Other F1 embryos (<i>m/+</i>) displayed HRMA profiles indicating heterozygosity for a newly induced mutant allele (red). Several distinct HRMA profiles were identified among the heterozygous F1 progeny of a single G0 founder, indicating multiple mutations were transmitted by each G0 founder.</p